Fluorescent calibration microbeads simulating stained cells

ABSTRACT

A method of calibrating a flow cytometer or fluorescent microscope is based on a set of highly uniform microbeads associated with a fluorescent dye in such a way that the microbeads have the same excitation and emission spectral properties as the samples which are to be measured. The calibration values of the microbeads are plotted against the relative fluorescence intensity peak channel for each microbead in the set. From this calibration plot, the relative fluorescence intensity peak channel of the sample is translated into equivalent soluble fluorescent dye molecules per sample particle. The calibration values of the standard microbeads are determined against solutions of the dyes. In cases where the background scatter of the bulk microbeads suspensions is too high for a direct determination against the solutions, a different set of microbeads with low background scatter is calibrated against the dye solutions and used to make an initial calibration of a flow cytometer or fluorescent microscope, which in turn, is used to calibrate the uniform microbead standards. A novel method of making the microbead standards is also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a division of co-pending application Ser. No. 805,654, filedDec. 11, 1985, which in turn is a continuation-in-part of co-pendingapplication Ser. No. 685,464, filed Dec. 24, 1984.

TECHNICAL FIELD

The invention relates to uniform, polymeric, fluorescent microbeads, thesynthesis thereof and their use in aligning and calibrating flowcytometry, fluorescent microscopes, and other instruments.

BACKGROUND ART AND REFERENCE DESCRIPTION

The majority of analytical cytology is conducted using highlysophisticated instruments such as fluorescence microscopes and flowcytometers to examine, count, or otherwise analyze cells stained withfluorescent dyes. Without fluorescent calibration standards, it is notpossible to make quantitative determinations about the cells withrespect to their fluorescence intensities.

With fluorescence microscopes, it would be useful to have fluorescentcalibrated particles which have the same size and fluorescent spectralproperties as the stained cells. Such particles could be used tooptimize the alignment of the microscope. If such particles hadcalibrated numbers of fluorescent molecules on them, they could be usedas an internal quantitative standard, i.e., mixed with the cells on aslide to compare the brightnesses of cells and particles. Quantitativeestimates of the fluorescence intensity of the cells could be made byeye, or accurate measurements could be made with photocells attached tothe fluorescent microscope.

Flow cytometry is the process of analyzing and sorting cells in aflowing stream. This is accomplished by intersecting the stream with anincident light, usually a laser, and detecting the resulting scatteredlight and fluorescence of the individual cells as a function of theparticular physical characteristics or attached fluorescent dye,respectively. In addition, electronic volume sensing has also beenincorporated in some flow cytometry instruments. With this array ofdetectors, sub-populations of cells can be analyzed and sorted inarbitrary terms by just detecting their qualitative differences.However, without size and fluorescent standards, no quantitativeinformation on the individual cells can be gained other than the numberof them counted and their proportion relative to the rest of the sample.

To determine quantitative differences between subpopulations of cells,and moreover, to give individual populations a quantitative relevance,standards are necessary with known amounts of fluorescence to whichthese cell samples can be compared. In FIG. 1, a microbead containing afluorescent dye, fluorescein isothiocynate (FITC), is shown along with acell labeled with the same dye. If a series of such microbeadscontaining varying amounts of the fluorescent dye is run on a flowcytometer, the resulting distributions will be obtained, as shown inFIG. 2, indicated by "Bead 1, Bead 2, and Bead 3". Now if a cellpopulation stained with the same dye is also run on the flow cytometerunder the same conditions, then the fluorescence intensity of the cellscan be quantitatively compared to those of the calibrated microbeads.

Various fluorescent particles have been used in conjunction with flowcytometry including fixed cells, pollen, fluorescent microbeads, andstained nuclei. However, their use has been limited for the most part toinstrument alignment and size calibration. Quantitation of fluorescenceintensity of cell samples has been hampered by not having a highlyuniform, stable particle which has the same excitation and emissionspectra as the cells being measured. Those particles which contain theproper dyes, e.g., the fluorescein stained nuclei (marketed asFluorotrol-GF by Ortho Diagnostic Systems, Inc.) are not stable overlong periods of time and those which are considered stable, e.g., themicrobeads, have not contained the same dyes which stain the cells.Highly uniform fluorescent microbeads have been available from varioussources for a number of years. However, none of these beads have beensuitable as quantitative standards for flow cytometry instrumentsbecause (1) many of these fluorescent microbeads are smaller than thecells to be analyzed and (2) the fluorescent dyes which have beenincorporated into the small microbeads are different from those attachedto the cells and (3) beads which are the preferable size (about 4-10microns in diameter) have been expensive to produce and difficult toobtain because the techniques used in their production give particles ofwide variability in size, shape, and other physical properties.

Attempts have been made to cross-calibrate microbeads containing one dyeagainst solutions or cells containing a different dye, e.g., cumerincontaining microbeads against fluorescein solutions. However, such acalibration is only good for the one excitation and emission filtersystem. Use of slightly different filter systems, which may occur withinstruments from different manufacturers, can significantly alter thequantitative results. As recognized in parent application Ser. No.685,464, the key to having a useful fluorescent standard which can beused on any instrument or filter system is for the microbead to have thesame excitation and emission spectra as the sample. A more subtle pointrecognized by the invention is that the environment of the dye moleculescan have a large effect on the fluorescence spectra. This isdemonstrated in FIG. 3, where the emission intensity of a cell labeledwith fluorescein is compared to that of microbeads with fluorescein onthe surface and fluorescein within the body of the hydrophobicmicrobead. The surface fluorescenated microbead has the fluorescein incontact with the aqueous medium, and has the same emission properties asa function of wavelength as does the fluorescein labeled cell suspendedin an aqueous medium, whereas, the microbead with the fluorescein withinthe hydrophobic bead body has a very different response because both theexcitation and emission spectra have shifted and broadened as a functionof the dye being in a hydrophobic medium and not in contact with water.The related spectra as recognized by the invention are shown in FIG. 4.

Recently, larger microbeads suitable for size calibration of biologicalcells have been synthesized in outer space (see NASA TM 78132"Large-size Monodisperse Latexes as a Commercial Space Product", andU.S. Pat. No. 4,247,434). In addition, U.S. Pat. No. 4,336,173 toUgelstad discloses a method for preparing uniform microbeads of a sizesuitable for flow cytometry calibration in a conventional, earth-boundlaboratory. However, none of these large microbeads were reported tocontain a fluorescent dye. Moreover, synthesis as described in U.S. Pat.No. 4,336,173 was found to be hampered by agglomeration and high doubletformation. Even with the suggested polymeric stabilizers, the yields ofmono-dispersed microbeads were lower than acceptable. The presentinvention has for its object, among others, the accomplishment of thistask.

In the Ugelstad method, a dispersion of small, uniform, seed particlesproduced by conventional means is contacted with a first swelling agentwhich is absorbed into the particles to cause some swelling of theparticles. Subsequently, a second swelling agent, usually apolymerizable monomer, is likewise absorbed into the particles. Uponinternal polymerization of this monomer, the particle undergoesadditional swelling. The desired particle size is reached by absorbingpre-calculated amounts of the first and second substances into themicroparticle.

Ugelstad teaches that, when using an oil soluble polymerizationinitiator which is somewhat soluble in water, the initiator may be addedafter the monomer has diffused into the particles or it may be dissolvedin the monomer before the latter diffuses into the particles (seeUgelstad; at column 6, line 65 to column 7, line 2). Ugelstad furtherteaches that, when using oil soluble initiators which are less watersoluble, it will be necessary to add the initiator together with thefirst swelling agent (see column 7, lines 2-5). When manufacturingbeads, however, adding the initiator with the first swelling agent isdisadvantageous because the initiator is unstable and swelled beadscontaining it must be handled carefully and used quickly. By notincluding the initiator in the first swelling agent the manufacturer canmake a large stable batch of swelled seed beads which can be stored foryears, giving the manufacturer the ability to choose the initiator andsecond swelling agent flexibly while using the same swelled seeds, andenabling the manufacturer to get more reproducible batches of finishedpolymerized beads by allowing him to draw from a uniform stock solutionof swelled beads. At the same time, however, it is desirable to use lesswater soluble initiators while manufacturing beads, as such initiatorsare less likely to cause agglutination among the beads and decrease theyield of monodisperse beads. In other words, the prior art teaches that,in order to get good yields of monodisperse beads, an initiator having avery low solubility in water should be used. However, the prior artfurther teaches that, when such an initiator is used, it should be addedwith the first swelling agent, and this requirement deprives themanufacturer of the advantages set forth above.

It is therefore an object of this invention to provide a method ofmanufacturing relatively large polymeric microbeads so that there is avery high yield of monodisperse beads without requiring the manufacturerto add the initiator to the seed beads at the same time the firstswelling agent is added. Other objects will appear as the descriptionproceeds.

DISCLOSURE OF INVENTION

The present invention is based on the synthesis and calibration of a setof highly-uniform, polymeric microbead standards. The microbeadstandards are used to align and calibrate fluorescent microscopes andflow cytometers over the size and fluorescence range of stained cells.The fluorescent microbeads of the invention exhibit excitation andemission spectra equivalent to those of the samples being measured. Thecalibration of fluorescence intensity of the microbeads is in terms ofnumber of equivalent soluble fluorescent molecules per microbead.

DESCRIPTION OF DRAWINGS

FIG. 1 schematically illustrates a microbead containing a fluorescentdye compared with a cell labelled with the same dye.

FIG. 2 illustrates the fluorescent intensity distribution of severalmicrobeads run on a flow cytometer.

FIG. 3 illustrates a comparison of emission intensity of a fluoresceinlabelled all with microbeads having fluorescein on the surface and withother microbeads having fluorescein within the body of the microbead.

FIG. 4 compares the spectra of fluorescein loaded microbeads in anaqueous medium compared with being in a non-aqueous medium.

FIG. 5 is a block diagram illustrating the steps for synthesizing themicrobeads of the invention.

FIG. 6 is a block diagram of the steps involved in calibrating thefluorescent microbeads of the invention.

FIG. 7 is a block diagram illustrating calibration of an instrumentutilizing the invention microbeads.

FIG. 8 illustrates microbead surface fluorescenation.

FIG. 9 is a dot plot of a set of five calibration microbeads withvarying size and fluorescence intensities.

FIG. 10 is a plot on logarithmic scale of Equivalent Soluble FluorescentMolecules versus Relative Fluorescent Channel.

BEST MODE FOR CARRYING OUT THE INVENTION

The synthesis of large-sized microbeads (1-20 microns, preferably 3-9microns in diameter) is accomplished according to the invention byswelling seed microbeads with two or more substances. The swelling issuch that as prescribed by thermodynamics, the enthropy of mixing withinthe seed microbead allows the seed to swell many times over the amountthe seed would swell if mixed with a single substance. While thisgeneral approach has been previously described in U.S. Pat. No.4,336,173, further improvements are critically needed with regard to thestability of the systems and examples described in such patent.Microbeads with less tendency to agglomerate and with less tendency toform doublets are needed. The present invention thus seeks to provide amore satisfactory stability and other improved characteristics in themicrobeads.

In the present invention, high concentrations of the oil solubleinitiator are dissolved in the oil soluble monomers. This solution isthen homogenized with an aqueous surfactant solution prior to using itto swell the microbeads already containing an oil soluble substance. Inaddition, the invention system is stabilized with alkaline halide salts,preferably potassium chloride, to keep the seeds separated duringswelling and polymerization. Once swollen, the suspension is purged withnitrogen and heated to initiate polymerization. It was discovered thatthe high concentrations of oil soluble initiator (0.5-5%) caused suchrapid polymerization within the swollen seeds, that polymerization ofmonomer in the aqueous phase could not proceed far enough to causeagglutination. It was further observed that any monomer/initiatordroplets which were not taken up by the seeds would polymerizeindividually, since they contained initiator, forming a second smallerpopulation of microbeads and again avoiding agglutination of the othermicrobeads, especially since the system was stabilized by the additionof salt containing large cations, e.g., potassium. Yields of thelarge-sized monodispersed microbeads were above 95 percent.

More specifically, the invention method is directed to making microbeadsof highly uniform and pre-determined size. The microbeads are preparedfrom a dispersion of polymeric microparticles, hereinafter referred toas seed particles, which are highly uniform in size but substantiallysmaller than the size of a cell. The dispersion of seed particles iscontacted with a first swelling agent, which should be a substance ofone or more materials having a molecular weight of less than 1000 and awater solubility of less than 10⁻³ g/l. The preferred first swellingagent is 1-chlorododecane. Other first swelling agents could be selectedfrom the group consisting of cetyl alcohol, 1-chlorononane,1-bromododecane and 1-dodecanol. Seed beads may be formed of polyvinylchloride, polyvinyl toluene, styrene, or methylmethacrylate withpolyvinyl toluene preferred.

The general guidelines provided by U.S. Pat. No. 4,336,173 areconsidered helpful in practicing the present invention and areincorporated herein by reference. Important distinctions between thematerial being incorporated herein and the method of the presentinvention are later indicated.

The second swelling agent of the invention will always be comprised ofone or more polymerizable monomers. If the bead is to be madefluorescent or have some other material connected to its surface, it isdesirable to have an ethylenically unsaturated compound having athree-membered epoxy ring as one of the monomers. Representative epoxymonomers include unsaturated alkyl glycidyl esters, unsaturated alkylglycidyl ethers, unsaturated cycloalkyl glycidyl ethers, unsaturatedalkyl-substituted phenyl glycidyl ethers, and the monoepoxide compoundsof the diene type monomers.

Suitable glycidyl esters include glycidyl methacrylate, glycidylacrylate, glycidyl esters of crotonic acid and long chain unsaturatedfatty acids and the like; unsaturated alkyl glycidyl ethers includevinyl glycidyl ether, isopropenyl glycidyl ether, oleyl glycidyl ether,allyl and methallyl glycidyl ethers and the like, unsaturated cycloalkyland phenyl glycidyl ethers include 4-vinyl cyclohexyl glycidyl ether,p-vinylbenzyl glycidyl ether, o-allyl phenylglycidyl ether, and thelike; the monoepoxide compounds of the diene type monomers includebutadiene monoepoxide, chloroprene monoepoxide, 3,4-epoxy-1-pentene,4,5-epoxy-1-hexene, 3,4-epoxy-1-vinylcyclohexene and divinylbenzenemonoxide and the like. The preferred monomer of the foregoing is methylmethacrylate. Second string preferred monomers comprise the groupconsisting of sytrene, vinyl tolulene, ethyl methacrylate, ethylacrylate and methyl acrylate.

The monomer having an epoxy ring can be used alone, but is, in thepreferred embodiment, combined with a second copolymerizable monomer toform the second swelling agent. This second monomer should becopolymerizable with the other monomer being used to form the secondswelling agent. Suitable monomers for this purpose include themonovinylidene carbocyclic monomers, e.g., styrene, alpha-methylstyrene,ar-(t-butyl)styrene, ar-methylstyrene, ar,ar-dimethylstyrene,ar-chlorostyrene, ar-(t-amyl)styrene, ar-bromostyrene, ar-fluorostyrene,ar-cyanostyrene, ar-methoxystyrene, ar-ethylstyrene,ar-hydroxymethylstyrene, ar-ethoxystyrene, a-chloro-ar-methylstyrene,ar,ar-dichlorostyrene, ar,ar-difluorostyrene, vinyl naphthalene, andother such emulsion polymerizable monomers having no more than 26 carbonatoms; esters of alpha,beta-ethylenically unsaturated carboxylic acidswhich polymerize to form non-film forming polymers, e.g., methylmethacrylate, chloroethyl methacrylate, n-butyl methacrylate, ethylmethacrylate, isobutyl methacrylate, isopropyl methacrylate, phenylmethacrylate, butyl chloroacrylate, cyclohexyl chloroacrylate, ethylchloroacrylate, methyl chloroacrylate, isopropyl chloroacrylate andother such esters capable of being polymerized to form hard polymers;alpha,beta-ethylenically unsaturated esters of non-polymerizablecarboxylic acids, e.g., vinyl benzoate, vinyl toluate, vinylar-ethylbenzoate, allyl ar-ethylbenzoate, vinyl trimethylacetate, vinylpivalate, vinyl trichloroacetate and other such monomers wherein theunsaturated moity has from 2 to 14 carbon atoms and the acid moity hasfrom 2 to 12 carbon atoms; alpha,beta-ethylenically unsaturatednitriles, e.g., such as nitriles having not more than 12 carbon atoms,other polymerizable vinyl monomers such as vinyl chloride, vinyl bromideand the like. Of the foregoing, the preferred monomer, to be used incombination with glycidyl methacrylate to form the second swellingagent, is methyl methacrylate. The copolymerization parameters r₁ r₂should match as closely as possible and stil retain the solubilityconsiderations, i.e., the second swelling should be more soluble inwater than first swelling agent but still be less than 10⁻³ molarity inwater. Second string monomers other than those mentioned can includethose containing a carboxyl group that can be activated by carbodiimide,e.g., methacrylic acid, a primary amine group which can be activated byaldehydes, e.g., ally amine, or an isiothiocynate group which is selfreactive at alkaline Ph.

In the present invention, high concentrations of an oil solubleinitiator, 0.5 to 5% by volume, are dissolved in the second swellingagent. This solution is then homogenized with an aqueous surfactantsolution prior to using it to swell the microbeads already containing afirst swelling agent. Homogenization aids in rapid incorporation of themonomer/initiator solution into the microbead seeds. It is preferred,but not essential, to add a stablizing agent to the dispersion ofswelled seed particles before the addition of the second swelling agent.A preferred stabilizing agent is a salt such as solution of an alkalimetal halide in an aqueous solution of the emulsifying agent used forthe swelling agent. Among the salts which may be used are the chlorides,bromides and iodides of potassium and cesium, with potassium chloridebeing preferred. The amount of halide salts which may be used is frombaout 0.001M-0.1M. Alkali metals forming hydrated ions smaller thanpotassium, such as sodium and lithium, are less effective as stabilizingagents.

Once swollen, the suspension is purged with nitrogen and heated toinitiate polymerization. It was discovered that the high concentrationsof oil soluble initiator (0.5-5%) caused such rapid polymerizationwithin the swollen seeds, that polymerization of monomer in the aqueousphase could not proceed far enough to cause agglutination. It wasfurther observed that any monomer/initiator droplets which were nottaken up by the seeds would polymerize individually, since theycontained initiator, forming a second smaller population of microbeadsand again avoiding agglutination of the other microbeads, especiallysince the system was stabilized by the addition of salt. Yields of thelarge-sized monodispersed microbeads were approximately 95 percent.

It is crucial that the initiator chosen be soluble in the secondswelling agent, and that the solubility of the initiator in the secondswelling agent be greater than its solubility in water. Chemicalssuitable for use as swelling agents include benzoyl peroxide anddioctanlyl peroxide, with benzoyl peroxide preferred. Lauryl peroxidewas tried in the method of the invention, using methyl methacrylate andglycidyl methacrylate as the second swelling agent, but was found not asefficient. These solubility parameters insure that, prior topolymerization, the second swelling agent will be more likely to escapefrom the beads than initiator. Moreover, most of the initiator that doesescape will be contained in droplets of free monomer rather thandissolved in the aqueous phase, thus resulting in decreased free chainpolymerization and agglutination. As mentioned above, any droplets ofmonomer and initiator polymerize separately to form a smaller, easilyseparated, population of particles.

Microbead Fluorescenation

Although fluorescent dyes can be diffused into or copolymerized into theinvention microbead, such microbeads may not be useful as fluorescentstandards. This is so because incorporation of the dye into anon-aqueous environment can cause spectral shifts in the dye. Suchspectral shifts would render the microbead near worthless asquantitative fluorescence standards. To be useful as a quantitativefluorescent standard, the microbeads have to have a functionality ontheir surface by which the fluorescent dye molecules can be attached.This arrangement maintains the fluorescent dye in the same aqueousenvironment as that of the cells labeled with the dye, thus retainingequivalent spectra. The preferred functional group on the surface of thelarge microbead is the epoxy group. It has versatility, in that it canbe directly activated under mild conditions, pH 8.5-10, to covalentlylink with amine-containing dyes, e.g., fluorescein amine orphycoeyrthrine, or it can be linked to a spacer group, e.g.,1,3-diaminopropane or 1,6-diaminohexame which in turn can be linked to areactive dye, e.g. fluorescein isothiocynate (FITC), under similar mildconditions, pH 8.5-10, to ensure that the dye is surrounded by theaqueous medium. This scheme is illustrated in FIG. 8.

Calibration of the Fluorescent Microbead Standards

Some researchers have attempted to calibrate fluorescent microbeads aswell as proteins with radioactive labels in terms of absolute numbers ofdye molecules. This approach results in a quagmire of correction factorsinvolving quenching considerations and change in extinction coefficientsdue to the chemical conjugation of the dye. These problems are reflectedby the fact that there are as of yet no NBS accepted primary fluorescentstandards for quantitative intensity, let alone for those specificfluorescent dyes of interest in flow cytometry.

Although the idea of quantitation in terms of absolute numbers ofmolecules of a fluorescent dye is attractive, its practicality at thistime is unobtainable. Therefore, an alternative calibration system isprovided by means of the present invention which relate a microbeadstandard back to a stable and reproducible solution of primary standardwhich has the same excitation and emission spectra as the sample beingmeasured. With sufficiently dilute solutions, considerations ofquenching and changes of extinction coefficient may be avoided, as longas the spectra of the primary soluble standard solution, the microbeadstandards, and the labeled cells in the sample are the same. Thus,fluorescent intensities of a sample may be related to a quantitativeconcentration of a soluble primary standard via calibrated microbeadswhich have the same spectra.

The fluorescenated microbeads are calibrated with such a system in termsof Equivalent Soluble Dye Molecules per Microbead. For example,fluorescein microbeads are standardized against a primary laser gradefluorescein. Laser grade fluorescein was chosen because it is the moststable, and of the highest purity, of any of the fluorescein compounds.Also, it has excitation and emission spectra equivalent to that ofFITC-labeled cells and the fluorescein microbead standards.

The fluorescent microbead standards are calibrated by determining thefluorescent intensity of standard solutions of laser grade dyes with afluorometer and relating those fluorescence intensities to thefluorescence intensity of suspensions of the microbeads. The number ofmicrobeads in the suspension per unit volume is determined with aCoulter Counter™ or a Hemocytometer™. Then from these data, the numberof equivalent soluble dye molecules per microbead is calculated bydividing the equivalent soluble dye molecules per unit volume by thenumber of microbeads per that unit volume. In the actual practice ofcalibrating the fluorescent microbead standards, the refractive index ofthe blank beads (those unfluorescenated) is too high which causes highbackground scatter (250,000 equivalent soluble dye molecules permicrobead). This makes it impossible to directly calibrate them in bulksuspension in a fluorometer. In this case, a different primary microbeadwhose scatter from the blank microbead is low (1000 equivalent solubledye molecules per microbead) must be used for calibration against thedye solutions. Such primary microbeads may be synthesized fromhydrophillic monomers as 2-hydroxy ethyl methacrylate, methacrylic acidacrylamide, and allyl fluorescein as described in U.S. Pat. Nos.4,157,323; 4,285,819 and 4,326,008. These primary microbeads are thenused to calibrate the flourescence channels of a flow cytometer, and inturn the original flourescent microbead standards are calibrated againstthe plot developed with the primary microbeads as shown in FIG. 10.These primary microbeads are themselves not as useful as the standardmicrobeads because they are too small (0.5-1.0 microns), and are notvery uniform.

Synthesis of Microbeads EXAMPLE 1

One milliliter of 1-chlorododecane (CDD) was homogenized with 2.5 ml of0.25% sodium dodecyl sulfate (SDS) in water and this was added to 5 mlof 10% suspension of 2.02 micron polyvinyl toluene microbeads in 20 mlof SDS solution. Ten milliliters of 30% acetone in water was added tohelp incorporate the CDD into the microbeads. This was stirred for 12hours before 1 ml of the suspension was added to 10 ml of distilledwater and 20 ml of SDS and evacuated to remove the acetone. Two hundredmilligrams (4%) of benzoyl peroxide initiator was dissolved in a 5 mlsolution of 95% methyl methacrylate and 5% glycidyl methacrylate beforeit was homogenized with an equal volume of 0.25% SDS solution. The tenmilliliters of the homogenate was then added to the above evacuatedsuspension of swollen seed microbeads and the suspension was purged withnitrogen and heated to 70° C. for two hours to cause rapidpolymerization of the swollen microbeads. The result was a highlyuniform microbead with a diameter of 5.3 microns. The yield was 96percent.

EXAMPLE 2

The procedure was the same as in Example 1, with the exception that 20ml of homogenized monomers and initiator was added to the seedsuspension resulting in microbeads 8.7 microns in diameter.

EXAMPLE 3

The procedure was the same as in Example 1, with the exception that 100milligrams (2%) of benzoyl peroxide initiator was used. The results werethe same as in Example 1.

EXAMPLE 4

The procedure of Example 3 was repeated except 25 mg. of benzoylperoxide initiator (0.5%) was used. There was increased aggregation ofthe microbeads indicating 0.5% to be a practical lower limit ofinitiator concentration. There was a yield of approximately 85%.

Fluorescenation of the Microbeads EXAMPLE 5

The microbeads in Examples 1 and 2 were washed in 0.25% SDS solution andto them was added an equal volume of 10% 1.3-diaminopropane adjusted topH 10.0. This was stirred for 12 hours then washed in SDS solution threetimes and in 0.1M NaHCO₃, pH 8.5 two times. FITC was added to portionsof these aminated suspensions and then they were washed four times in0.05 M phosphate buffer pH 7.2. This resulted in green fluorescentmicrobeads as viewed in an epiluminescent fluorescent microscope usingblue exciting light.

EXAMPLE 6

The same procedures was used as in Example 5, except Texas Red was usedto replace FITC. The result was red fluorescent microbeads as viewedunder the fluorescence microscope using green exciting light.

EXAMPLE 7

The microbeads obtained in Examples 1 and 2, still containing functionalepoxy groups, were mixed with a solution of phycoerythrin at a pH of 9.5for 12 hours. After washing the resulting microbead had an orangefluorescence under the fluorescence microscope.

EXAMPLE 8

The same procedure was carried out as in Example 7, except themicrobeads were added to a solution of avidin. Following washing in PBSpH 7.2, the microbeads coated with avidin were exposed to a solution ofbiotin-phycoerythrin and the microbeads had an orange fluoresence underthe fluorescence microscope.

EXAMPLE 9

The microbeads in Example 5 were fluorescenated at predetermined levelsof fluorescence intensity with FITC by introducing small amounts of thedyes stepwise into the microbead suspensions and checking the microbeadsfluorescence intensity with a flow cytometer. Specifically, 1 mg of FITCwas dissolved in 3 ml of methanol and this was added dropwise to asuspension of microbeads from Example 1 in 0.1M NaHCO₃ at pH 8.5 whileintermittently determining the fluorescence level with a flow cytometer.Only enough FITC methanol solution was added to the microbeads to reachthe desired microbead fluorescence intensity. Other suspensions of thesame microbeads were brought to different levels of intensity resultingin a set of microbeads of various specific predetermined fluorescencelevels, i.e., 2×10⁴,4×10⁵, 9×10⁵ and 2×10⁶, which an be used to developa calibration curve for a flow cytometer as seen in FIGS. 9 and 10.

Calibration EXAMPLE 10 Step 1

Excitation and emission spectra were taken in PBS pH 7.2 of solublelaser grade fluorescein, hydrophilic (low background scatter) 1 micronmicrobeads, and the microbeads from Example 6. All the excitationspectra of these samples had a peak at 293 (matched within 3 nanometers)and had equivalent shapes. They also had matching emission spectra withthe peak at 518 nanometers.

Step 2

In 1000 mls. of PBS pH 7.2, 9.6 mgs of laser grade fluorescein wasdissolved and further diluted 1:100 making a solution of 4.8×10⁻⁸ Mfluorescein. The fluorescence intensity of this solution was read in afluorometer and then ratioed against the readings of dilute microbeadsuspensions (1 million microbeads per ml) to determine the equivalentfluorescein molarity of the microbead suspensions. This molarity wasdivided by the number of microbeads per ml (1.4 million per ml) asdetermined with a Hemocytometer. This resulted in 7.8×10⁵ solublefluorescein molecules per microbead.

Step 3

A set of the small calibrated hydrophilic microbeads were run on a flowcytometer and a calibration plot was made of the microbead fluorescenceintensity versus the instrument fluorescent channel number. Thiscalibration curve was then used in turn to quantitate the fluorescenceintensity in terms of equivalent soluble fluorescein molecules permicrobead of the set of microbeads in Example 9.

Use of the Microbeads EXAMPLE 11

A set of four fluorescent calibrated microbeads as prepared in Example9, designated 0(blank), +1(2×10⁴), +2(9×10⁵), +3(2×10⁶), were used witha fluorescence microscope by mixing them with the stained cell samplesand comparing them in intensity by eye or with a photocell to thestained cells. The designations 0, +1, +2, +3 are equivalent to thearbitrary assignments given in clinical laboratories and thedesignations blank, 2×10⁴, 9×10⁵, 2×10⁶, are in terms of equivalentsoluble fluorescent molecules per microbead. As long as the microbeadsare the same size as the cells being examined, the quantitative estimateof numbers of equivalent soluble fluorescent molecules per cell asdetermined by comparison to the microbeads will be accurate. Lymphocytesstained with FITC-Leu 1 antibody appear to have the same fluorescenceintensity as the +1 microbead and taken to have 2×10⁴ equivalent solublefluorescein molecules per cell.

EXAMPLE 12

A FACS Analyzer™ (flow cytometer) was calibrated with the microbeads inExample 9. The cells in Example 11 were run on the FACS Analyzer™without changing any of the instrument settings. This resulted in havingthe cells fall in a relative intensity channel equal to 2.5×10⁴equivalent soluble fluorescein molecules per cell.

SUMMARY

The invention in its various aspects provides a method by which afluorescent microscope or flow cytometer may be calibrated in terms ofthe number of equivalent soluble fluorescent dye molecules perfluorescence intensity channel of the instrument by the use of highlyuniform microbeads with a fluorescent dye associated therewith, suchthat the microbeads have the same excitation and emission spectra assamples being measured. The invention method is thus based on themicrobeads themselves being calibrated in terms of equivalent numbers ofsoluble fluorescent dye molecules per microbead. Stated differently, theinvention method of calibrating the microbeads is based on determiningthe number of equivalent soluble fluorescent dye molecules necessary togive rise to the same level of fluorescence intensity as the particularmicrobead. This is accomplished by determining the fluorescenceintensity of a suspension of microbeads with a fluorometer as comparedto solutions of the free fluorescent dye, and dividing by the number ofmicrobeads in the suspension to yield the number of equivalent solublefluorescent dye molecules per microbead. When background scatter ofthese microbeads is too high for direct calibration, a second type oflow background scatter hydrophilic microbead is calibrated against thedye solutions and these low background scatter microbeads are thencalibrated against the hydrophilic microbeads.

The calibration microbeads useful for the invention have the followingproperties:

(a) They are highly uniform and in the size range of the sample cellswhich are being measured, 1-20u in diameter, preferably 3-9u indiameter.

(b) While primarily directed to animal cells ranging from 1-20u indiameter, those skilled in the art will immediately recognize that thisinvention is applicable to calibration of larger 60-1OOu cells, such aschloroplasts found in plants.

(c) They have associated with them a fluorescent dye that will give riseto the same excitation and emission spectra as that of the cell samplewhich is being measured.

(d) They have fluorescence intensities between 10³ -10⁷ equivalentsoluble molecules of fluorescent dye per microbead.

(e) They are stable with respect to size and fluorescence intensity intheir suspending media, which in turn is the same as that in which thecell samples are suspended.

The calibration microbeads are composed of hydrophobic polymericmaterials which have chemically functional groups on the surface of themicrobeads such that a fluorescent dye may be conjugated via a stablebond, and such that this bonding will maintain the fluorescent dye incontact with the suspension medium. The hydrophobic polymeric materialsare preferably a co-polymer of 95% methyl methacrylate and 5% glycidylmethacrylate, but the composition could include co-polymers in variousratios of styrene, vinyl toluene, and other acrylate and methacrylateesters with glycidyl methacrylate, allyl glycidyl ether, or other epoxycontaining monomers.

The preferred method of making the calibration microbead standard is tofirst swell seed beads with an oil soluble compound, then following witha second swelling with a aqueous homogenate which contains a monomerwith a high concentration of oil soluble initiator (0.5-5%) dissolved init, which will cause polymerization at such a rapid rate in the oil(monomer) phase as to minimize any polymerization in the aqueous phase,thus reducing agglomeration of the microbeads.

The preferred linkage to the microbeads of the fluorescent dye isthrough a covalent linkage which can be generated by first animating thesurface of the microbeads with a diamine, preferably 1,3-diaminopropaneor 1,6-diaminohexane, through reacting with the epoxy surface group,then reacting the aminated surface of the microbeads with a reactivefluorescent dye, such as fluorescein isothiocynate or Texas Red.However, a stable linkage can be directly formed to the microbead viathe epoxy group reacting directly to a primary amine on the fluorescentdye, such as fluorescein amine or phycoerythrin.

Also to be noted is that sensitivity of the flow cytometer can bedetermined by using the microbead with the lowest level of fluorescenceand a blank microbead, i.e. a microbead without an attached fluorescentmolecule, in such a way as to determine the distance between fluorescentpeaks of the two microbeads being employed for such purpose.

From the foregoing, it can be seen that the invention not only providesa unique method for calibrating a flow cytometer but also a uniquesynthesis of the microbead standards related thereto.

What is claimed is:
 1. The method of making a calibration microbeadstandard to minimize aggregation and maximize yield comprising thesteps:(a) homogenizing a highly insoluble (<10⁻³ g/l) compound ofmolecular weight <1000 in a surfactant solution to obtain a firsthomogenate; (b) swelling small seed microbeads, 0.1-3u in diameter, withsaid first homogenate; (c) homogenizing an aqueous surfactant solutioncontaining a large stabilizing alkaline halide salt with a mixture ofoil soluble polymerizable monomers within which is dissolved 0.5-5% byweight an oil soluble initiator to obtain a second homogenate; (d)swelling the seed microbeads a second time with said second homogenate;and (e) placing said twice swollen microbeads under conditionsappropriate to cause said initiator to polymerize the monomers in theswollen microbeads.
 2. The method of claim 6 wherein said highly watersoluble compound is 1-chlorododecane.
 3. The method of claim 6 whereinsaid stabilizing alkaline halide salt is potassium chloride, said oilsoluble monomers methyl methacrylate and glycidyl methacrylate, and saidoil soluble initiator is benzoyl peroxide.